Summary A label-free imaging technology is proposed for general cancer research, termed as Optical Fiber-Tethered Simultaneous Lifetime-resolved Autofluorescence and Multiharmonic (OFT-SLAM) microscopy, to overcome the lack of a versatile tool to simultaneously visualize tumor and non-tumor cells in authentic tumor microenvironment. The non-tumor cells broadly include the fibroblastic cells, angiogenic vascular cells, and infiltrating immune cells that engage normal biological functions such as embryonic/adult development and inflammatory/immune response (e.g. wound healing). However, in the tumor microenvironment where the overall metabolism is known to switch from energy consumption to proliferative biosynthesis (the Warburg effect), these normal (neutral) cells have all been recently recognized as the accessories to the crime (cancer). Thus, the proposed development of this imaging technology will interrogate the interrelations between metastatic tumor cells (principal) and various non-tumor cells (accessories) that conspire to kill a cancer patient (crime). This interrogation will be more comprehensive than imaging-based cancer research that has typically focused on one specific cell type of interest (the principal or one accessory of the crime). Without a label-free imaging technology like OFT-SLAM to avoid cell-specific labeling, simultaneous visualization of various cells would perturb the tumor microenvironment by exogenous staining, cell/tissue transplantation, and genetic modification. We will build the ?SLAM? of OFT-SLAM based on multimodal multiphoton microscopy and fluorescence-lifetime imaging, and invoke general intrinsic contrasts of cellular optical heterogeneity and metabolic activity to reveal and differentiate tumor and non-tumor cells. We will then empower the ?SLAM? with the ?OFT? to flexibly access different anatomical sites in intravital animal/preclinical microscopy and ex vivo human/clinical histopathology. We will subsequently employ the resulting OFT-SLAM to image the formalin-fixed human specimens of breast cancer from Cooperative Human Tissue Network (CHTN), including the primary breast tumors, breast cancer- induced lung and brain metastases, and surrounding peri-tumoral fields at different stages from different patients (n > 200). In parallel, we will apply OFT-SLAM to long-term (imaging window-assisted) intravital microscopy of three prototypical breast cancer rat/mouse models, covering all known steps throughout the invasion-metastasis cascade. With the unique capability of OFT-SLAM to bridge otherwise isolated ex vivo human histopathology (snapshots taken by pathologists in a clinical setting) and intravital animal microscopy (movies acquired by biologists in a laboratory), we will strive to identify various cancer-associated cells and their interrelations in an evolving tumor microenvironment and their dependence on spatial heterogeneity and individual variability. The successful outcome of this project will demonstrate a versatile visualization tool to interrogate tumor microenvironment with built-in translational ability, and thus transform cancer diagnosis and therapy.